The present invention relates generally to blood products, and more particularly to compositions comprising mixtures of oxygen-carrying and non-oxygen carrying plasma expanders and methods for their use.
A. The Circulatory System and The Nature of Hemoglobin
The blood is the means for delivering nutrients to the tissues and removing waste products from the tissues for excretion. The blood is composed of plasma in which red blood cells (RBCs or erythrocytes), white blood cells (WBCs), and platelets are suspended. Red blood cells comprise approximately 99% of the cells in blood, and their principal function is the transport of oxygen to the tissues and the removal of carbon dioxide therefrom.
The left ventricle of the heart pumps the blood through the arteries and the smaller arterioles of the circulatory system. The blood then enters the capillaries, where the majority of the exchange of nutrients and cellular waste products occurs. (See, e.g., A. C. Guyton, Human Physiology and Mechanisms of Disease (3rd. ed.; W. B. Saunders Co., Philadelphia, Pa.), pp. 228-229 [1982]). Thereafter, the blood travels through the venules and veins in its return to the right atrium of the heart. Though the blood that returns to the heart is oxygen-poor compared to that which is pumped from the heart, in resting man the returning blood still contains about 75% of the original oxygen content.
The reversible oxygenation function (i.e., the delivery of oxygen and the removal of carbon dioxide) of RBCs is carried out by the protein hemoglobin. In mammals, hemoglobin has a molecular weight of approximately 68,000 and is composed of about 6% heme and 94% globin. In its native form, it contains two pairs of subunits (i.e., it is a tetramer), each containing a heme group and a globin polypeptide chain. In aqueous solution, hemoglobin is present in equilibrium between the tetrameric (MW 68,000) and dimeric forms (MW 34,000); outside of the RBC, the dimers are prematurely excreted by the kidney (plasma half-life of approximately two to four hours). Along with hemoglobin, RBCs contain stroma (the RBC membrane), which comprises proteins, cholesterol, and phospholipids.
B. Exogenous Blood Products
Due to the demand for blood products in hospitals and other settings, extensive research has been directed at the development of blood substitutes and plasma expanders. A blood substitute is a blood product that is capable of carrying and supplying oxygen to the tissues. Blood substitutes have a number of uses, including replacing blood lost during surgical procedures and following acute hemorrhage, and for resuscitation procedures following traumatic injury. Plasma expanders are blood products that are administered into the vascular system but are typically not capable of carrying oxygen. Plasma expanders can be used, for example, for replacing plasma lost from burns, to treat volume deficiency shock, and to effect hemodilution (for, e.g., the maintenance of normovolemia and to lower blood viscosity). Essentially, blood products can be used for these purposes or any purpose in which banked blood is currently administered to patients. (See, e.g., U.S. Pat. Nos. 4,001,401 to Bonson et al. and 4,061,736 to Morris et al., hereby incorporated by reference).
The current human blood supply is associated with several limitations that can be alleviated through the use of an exogenous blood product. To illustrate, the widespread availability of safe and effective blood substitutes would reduce the need for banked (allogeneic) blood. Moreover, such blood substitutes would allow the immediate infusion of a resuscitation solution following traumatic injury without regard to cross-matching (as is required for blood), thereby saving valuable time in resupplying oxygen to isclehleic tissue. Likewise, blood substitutes can be administered to patients prior to surgery, allowing removal of autologous blood from the patients which could be returned later in the procedure, if needed, or after surgery. Thus, the use of exogenous blood products not only protects patients from exposure to non-autologous (allogeneic) blood, it conserves either autologous or allogeneic (banked, crossmatched) blood for its optimal use.
C. Limitations of Current Blood Substitutes
Attempts to produce blood substitutes (sometimes referred to as xe2x80x9coxygen-carrying plasma expandersxe2x80x9d) have thus far produced products with marginal efficacy or whose manufacture is tedious and expensive, or both. Frequently, the cost of manufacturing such products is so high that it effectively precludes the widespread use of the products, particularly in those markets where the greatest need exists (e.g., emerging third-world economies).
The blood substitutes that have been developed previously are reviewed in various references (See e.g., Winslow, Robert M., xe2x80x9cHemoglobin-based Red Cell Substitutes, xe2x80x9cJohns Hopkins University Press, Baltimore [1992]). They can be grouped into the following three categories: i) perfluorocarbon-based emulsions, ii) liposomexe2x80x94encapsulated hemoglobin, and iii) modified cell-free hemoglobin. As discussed below, none has been entirely successful, though products comprising modified cell-free hemoglobin are thought to be the most promising. Perfluorochemical-based compositions dissolve oxygen as opposed to binding it as a chelate. In order to be used in biological systems, the perfluorochemical must be emulsified with a lipid, typically egg-yolk phospholipid. Though the perfluorocarbon emulsions are inexpensive to manufacture, they do not carry sufficient oxygen at clinically tolerated doses to be effective. Conversely, while liposomexe2x80x94encapsulated hemoglobin has been shown to be effective, it is far too costly for widespread use (See e.g.,Winslow, supra).
Most of the blood substitute products in clinical trials today are based on modified hemoglobin. These products, frequently referred to as hemoglobin-based oxygen carriers (HBOCs), generally comprise a homogeneous aqueous solution of a chemically-modified hemoglobin, essentially free from other red cell residues (stroma). Although stroma-free human hemoglobin is the most common raw material for preparing a HBOC, other sources of hemoglobin have also been used. For example, hemoglobin can be obtained or derived from animal blood (e.g., bovine hemoglobin) or from bacteria or yeast or transgenic animals molecularly altered to produce a desired hemoglobin product. (See generally, Winslow, supra).
The chemical modification is generally one of intramolecular crosslinking and/or oligomerization to modify the hemoglobin such that its persistence in the circulation is prolonged relative to that of unmodified hemoglobin, and its oxygen binding properties are similar to those of blood. Intramolecular crosslinking chemically binds together subunits of the tetrameric hemoglobin unit to prevent the formation of dimers which, as previously indicated, are prematurely excreted. (See, e.g., U.S. Pat. No. 5,296,465 to Rausch et al., hereby incorporated by reference).
The high costs of manufacturing HBOC products have greatly limited their commercial viability. In addition, the present inventors have found that known HBOCs have a tendency to release excessive amounts of oxygen to the tissues at the arteriole walls rather than the capillaries; this can result in insufficient oxygen available for delivery by the HBOC to the tissues surrounding the capillaries. This is despite the fact that the initial loading of the HBOC with oxygen may be relatively high, even higher than that normally achieved with natural red blood cells.
What is needed is a blood product that is relatively inexpensive to manufacture and that delivers adequate amounts of oxygen to the tissues.
The present invention is directed at compositions comprising mixtures of an oxygen-carrying component and a non-oxygen carrying component and methods for their use. The compositions overcome the limited oxygen delivery characteristics of previous blood substitutes, and therefore lower doses may be used. They are a safer and more effective alternative to currently available blood substitutes.
The present invention contemplates a means of improving the oxygen delivering capacity of an oxygen carrier by combining that carrier with a non-oxygen-carrying component like a conventional plasma expander. In preferred embodiments, the oxygen carrier (i.e., the oxygen-carrying component) is a hemoglobin-based oxygen carrier. The hemoglobin may be either native (unmodified); subsequently modified by a chemical reaction such as cross-linking, polymerization, or the addition of chemical groups (i.e., polyethyleneglycol, polyoxyethylene, or other adducts); or it may be recombinant or encapsulated in a liposome. A non-oxygen-carrying plasma expander is any substance used for temporary replacement of red cells which has oncotic pressure (e.g., starches such as hetastarch or pentastarch, dextran such as dextran-70 or dextran-90, albumin, or any other colloidal intravenous solution).
More specifically, it is contemplated that the compositions of the present invention will contain one or more of the following properties: i) viscosity at least half that of blood, ii) oncotic pressure higher than that of plasma; iii) hemoglobin oxygen affinity higher than or equal to (i.e., P50 equal to or lower than) that of blood; and iv) oxygen capacity less than that of blood. It is not intended that the invention be limited to how the compositions are used. A variety of uses are contemplated for the compositions of the present invention, including, but not limited to, the treatment of hemorrhage or use in hemodilution.
Particular non-oxygen carrying plasma expanders have been used (e.g., for hemodilution) for a number of years, and their physiological effects following administration are well characterized. Previously, researchers have assumed that administration of an oxygen-carrying blood product (e.g., a blood substitute like an HBOC), should result in physiological cardiovascular responses similar to those observed following administration of non-oxygen carrying diluent materials of similar molecular weight (e.g., dextran 70,000 MW, albumins and starches). Furthermore, researchers in the field of blood substitutes have been working under several other key assumptions. More specifically, prior to the present invention, it has been thought that blood substitutes should have viscosity less than that of blood, oxygen affinity similar to or equal to or lower than that of red cells, minimal colloidal osmotic (oncotic) pressure, and hemoglobin concentration as high as possible. As described in detail below, the compositions and methods of the present invention are counter-intuitive to some of these assumptions.
The present invention contemplates a blood product solution, comprising an oxygen-carrying component and a non-oxygen carrying component, the blood product solution having oncotic pressure higher than that of plasma and viscosity at least half that of blood. In some embodiments, the blood product solution further comprises oxygen affinity equal to or greater than that of blood. In other embodiments, the blood product solution further comprises oxygen capacity less than that of blood. In particular embodiments, the oxygen-carrying component is a polyethylene glycol-modified hemoglobin. Furthermore, in certain embodiments the non-oxygen-carrying component is a colloid starch. When the non-oxygen-carrying component is a colloid starch, it has an average molecular weight of from approximately 200,000 daltons to approximately 400,000 daltons is some embodiments. In particular embodiments, the colloid starch is pentastarch.
The present invention also contemplates a blood product solution, comprising a) an oxygen-carrying component, the oxygen-carrying component comprising a polyethylene glycol-modified hemoglobin; and b) a non-oxygen carrying component, the non-oxygen-carrying component comprising a colloid starch having an average molecular weight of from approximately 200,000 daltons to approximately 400,000 daltons. In some embodiments, the polyethylene glycol-modified hemoglobin comprises hemoglobin selected from the group consisting of animal hemoglobin, human hemoglobin, and recombinant hemoglobin. In particular embodiments, the colloid starch has an average molecular weight of from approximately 225,000 daltons to approximately 300,000 daltons, and in other embodiments the colloid starch is pentastarch. In still other embodiments, the pentastarch comprises from approximately 20 percent to approximately 80 percent by volume of the blood product solution, whereas the pentastarch comprises from approximately 40 percent to approximately 60 percent by volume of the blood product in other embodiments. Moreover, the blood product solution has a viscosity from approximately 2 centipoise to approximately 4.5 centipoise in particular embodiments.
The present invention also contemplates a method of enhancing oxygen delivery to the tissues of a mammal, comprising a) providing a blood product solution, comprising an oxygen-carrying component and a non-oxygen carrying component, the blood product solution having oncotic pressure higher than that of plasma and viscosity at least half that of blood; and b) administering the blood product solution to the mammal, thereby enhancing oxygen delivery to the tissues of the mammal. In some embodiments, the blood product solution further comprises oxygen affinity equal to or greater than that of blood, while in other embodiments the blood product solution further comprises oxygen capacity less than that of blood. In some embodiments, the oxygen-carrying component is a polyethylene glycol-modified hemoglobin. The non-oxygen-carrying component is a colloid starch in particular embodiments; in some embodiments, the colloid starch has an average molecular weight of from approximately 200,000 daltons to approximately 400,000 daltons. The colloid starch is pentastarch in still further embodiments.
In addition, the present invention contemplates a method of enhancing oxygen delivery to the tissues of a mammal, comprising a) providing a blood product solution, comprising i) an oxygen-carrying component, the oxygen-carrying component comprising a polyethylene glycol-modified hemoglobin, and ii) a non-oxygen carrying component, the non-oxygen carrying component comprising a colloid starch having an average molecular weight of from approximately 200,000 daltons to approximately 350,000 daltons; and b) administering the blood product solution to the mammal, thereby enhancing oxygen delivery to the tissues of the mammal.
In some embodiments, the polyethylene glycol-modified hemoglobin comprises hemoglobin selected from the group consisting of animal hemoglobin, human hemoglobin, and recombinant hemoglobin. In other embodiments, the colloid starch has an average molecular weight of from approximately 200,000 daltons to approximately 400,000 daltons. In still other embodiments, the colloid starch is pentastarch. In particular embodiments, the pentastarch comprises from approximately 20 percent to approximately 80 percent by volume of the blood product.
In certain embodiments, the blood product solution has a viscosity of from approximately 2 centipoise to approximately 4.5 centipoise. Finally, in other embodiments, the mammal is a human.
The present invention also provides an aqueous cell-free composition comprising hemoglobin, in which the hemoglobin is present in a concentration of between 0.1 and 4.0 g/dl, and the aqueous composition has a viscosity that is greater than 2.5 cP. In some preferred embodiments, the viscosity of the aqueous composition is between 2.5 and 4 cP. Thus, it is not intended that the present invention be limited to any viscosity that is greater than approximately 2.5 cP. Indeed, it is contemplated that the present invention encompass compositions in which the viscosity is 6 cP or greater. In addition, the present invention encompasses compositions in which the hemoglobin concentration is less than 0.1 or greater than 4 g/dl, although in particularly preferred embodiments, the hemoglobin concentration is between 0.1 and 4 g/dl. Furthermore, in some embodiments, the K* of the composition is approximately equal or similar to that of a red blood cell suspension when measured at the same hemoglobin concentration.
In other embodiments of the composition, the hemoglobin has an increased affinity for molecular oxygen as compared to red blood cells. The present invention provides compositions that are suitable for use in any animal, including humans. Thus, in some embodiments, the hemoglobin of the composition has an increased affinity as compared to mammalian red blood cells, although in other embodiments, it is contemplated that the red blood cells are from reptiles, avians, or any other animal. In most preferred embodiments, the red blood cells used in this comparison are human red blood cells. In preferred embodiments, the composition has a P50 of less than 28 mm Hg. However, it is not intended that the present invention be limited to this P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.
In other embodiments, the composition further comprises a diluent selected from the group consisting of proteins, glycoproteins, polysaccharides, and other colloids. It is not intended that these embodiments be limited to any particular diluent. Thus, it is intended that the diluent encompass solutions of albumin, other colloids, or other non-oxygen carrying components. In preferred embodiments, the diluent comprises polysaccharide. In other preferred embodiments, the polysaccharide comprises starch. In particularly preferred embodiments, the starch comprises pentastarch.
In other embodiments, the hemoglobin within the composition is surface-modified. It not intended that these embodiments be limited to any particular type of surface modification. In preferred embodiments, the surface modification includes the use of polyalkylene oxide groups of varying chain lengths and charges. In preferred embodiments, the hemoglobin is surface-modified with polyethylene glycol of varying chain lengths and charges. It is not intended that the surface modification be limited to any particular type or a single type of modification. It is contemplated, that multiple types of surface-modifications will be made to hemoglobin of the composition.
The present invention also provides an aqueous cell-free composition comprising surfaced-modified hemoglobin, wherein the surface-modified hemoglobin is present in a concentration of between 0.1 and 4.0 g/dl, and the aqueous composition has a viscosity that is greater than 2.5 cP. As discussed above, in some preferred embodiments, the viscosity of the aqueous composition is between 2.5 and 4 cP. Thus, it is not intended that the present invention be limited to any viscosity that is greater than approximately 2.5 cP. Indeed, it is contemplated that the present invention encompass compositions in which the viscosity is 6 cP or greater. In further embodiments, the hemoglobin concentration is less than 0.1 or greater than 4 g/dl, although in particularly preferred embodiments, the hemoglobin concentration is between 0.1 and 4 g/dl. In some embodiments, the K* of the composition is approximately equal or similar to that of a red blood cell suspension when measured at the same hemoglobin concentration.
In preferred embodiments of this composition, the hemoglobin has an increased affinity for molecular oxygen as compared to red blood cells. As above, these embodiments are suitable for use in any animal, including humans. Thus, in some embodiments, the hemoglobin has an increased affinity as compared to mammalian red blood cells, although in other embodiments, it is contemplated that the red blood cells are from reptiles, avians, or any other animal. In most preferred embodiments, the red blood cells used in this comparison are human red blood cells. In other preferred embodiments, the composition has a P50 of less than 28 mm Hg. In alternative preferred embodiments, the P50 is approximately 1, while in others, it is in the range of 1 and 1.5, while in still other embodiments, it is approximately 10. However, it is not intended that the present invention be limited to any particular P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.
Furthermore, in other embodiments, the present invention provides compositions which further comprise a diluent selected from the group consisting of proteins, glycoproteins, polysaccharides, and other colloids. It is not intended that the these embodiments be limited to any particular diluent. Thus, it is intended that the diluent encompass solutions of albumin, other colloids, or other non-oxygen carrying components. In preferred embodiments, the diluent comprises polysaccharide. In other preferred embodiments, the polysaccharide comprises starch. In particularly preferred embodiments, the starch comprises pentastarch. In these embodiments, it not intended that the present invention be limited to any particular type of surface modification. In preferred embodiments, the surface modification includes the use of polyalkylene oxide groups of varying chain lengths and charge. In preferred embodiments, the hemoglobin is surface-modified with polyethylene glycol of varying chain lengths and charges.
The present invention further provides an aqueous cell-free composition comprising a mixture of hemoglobin and a. diluent, wherein the hemoglobin is present in a concentration between 0.1 and 4 g/dl, and wherein the diluent is selected from the group consisting of proteins, glycoproteins, polysaccharides, and other colloids, and wherein the aqueous composition has a viscosity of at least 2.5 cP. As discussed above, in some preferred embodiments, the viscosity of the aqueous composition is between 2.5 and 4 cP. Thus, it is not intended that these embodiments be limited to any viscosity that is greater than approximately 2.5 cP. Indeed, it is contemplated that the present invention encompass compositions in which the viscosity is 6 cP or greater. Furthermore, in some embodiments, the diluent comprises a polysaccharide, while in preferred embodiments, the diluent comprises starch, and in particularly preferred embodiments, the diluent comprises pentastarch. In addition, the present invention encompasses compositions in which the hemoglobin concentration is less than 0.1 or greater than 4 g/dl, although in particularly preferred embodiments, the hemoglobin concentration is between 0.1 and 4g/dl. In some embodiments, the K* of the composition is approximately equal or similar to that of a red blood cell suspension when measured at the same hemoglobin concentration.
In other embodiments of the composition, the hemoglobin has an increased affinity for molecular oxygen as compared to red blood cells. The present invention provides compositions that are suitable for use in any animal, including humans. Thus, in some embodiments, the hemoglobin of the composition has an increased affinity as compared to mammalian red blood cells, although in other embodiments, it is contemplated that the red blood cells are from reptiles, avians, or any other animal. In most preferred embodiments, the red blood cells used in this comparison are human red blood cells. In preferred embodiments, the composition has a P50 of less than 28 mm Hg. In preferred embodiments, the composition has a P50 of less than 28 mm Hg. However, it is not intended that the present invention be limited to this P50 value, as in some embodiments, the P50 is higher.
As indicated above, these embodiments may also comprise hemoglobin that is surface-modified. It not intended that the present invention be limited to any particular type of surface modification. In preferred embodiments, the surface modification includes the use of polyalkylene oxide groups of varying chain lengths and charge. In preferred embodiments, the hemoglobin is surface-modified with polyethylene glycol of varying chain lengths and charges.
The present invention also provides methods comprising providing an animal and an aqueous cell-free composition comprising hemoglobin, wherein the hemoglobin is present in a concentration of between 0.1 and 4.0 g/dl, and the aqueous composition has a viscosity that is greater than 2.5 cP; and administering the aqueous composition to the animal. In preferred embodiments, the animal is a mammal, while in particularly preferred embodiments, the animal is human. In some embodiments, the human is suffering from the symptoms of disease, pathology, insufficiency, or abnormality. In some embodiments, the human has symptoms of disease, wherein the disease is selected from the group consisting of hypovolemic shock symptoms, hypoxia, chronic lung disease, ischemia, stroke, trauma, hemodilution, cardioplegia, cancer, anemia, sickle-cell anemia, septic shock, or disseminated intravascular coagulation. However, it is not intended that the methods of the present invention be limited to the administration of the aqueous composition to alleviate any particular disease, condition, pathology, insufficiency, or abnormality. Rather, it is intended that the methods encompass any and all applications for which the methods are suitable.
As above, the methods of present invention encompass an aqueous cell-free composition comprising hemoglobin, wherein the hemoglobin is present in a concentration of between 0.1 and 4.0 g/dl, and the aqueous composition has a viscosity that is greater than 2.5 cP. In some preferred embodiments, the viscosity of the aqueous composition is between 2.5 and 4 cP. Thus, it is not intended that the present invention be limited to any viscosity that is greater than approximately 2.5 cP. Indeed, it is contemplated that the present invention encompass compositions in which the viscosity is 6 cP or greater. In addition, the present invention encompasses compositions in which the hemoglobin concentration is less than 0.1 or greater than 4 g/dl, although in particularly preferred embodiments, the hemoglobin concentration is between 0.1 and 4 g/dl. In some embodiments, the K* of the composition is approximately equal or similar to that of a red blood cell suspension when measured at the same hemoglobin concentration.
In alternative embodiments, the compositions comprise hemoglobin with an increased affinity for molecular oxygen as compared to red blood cells. In addition, these embodiments are suitable for use with any animal, including humans. Thus, in some embodiments, hemoglobin has an increased affinity as compared to mammalian red blood cells, although in other embodiments, it is contemplated that the red blood cells are from reptiles, avians, or any other animal. In most preferred embodiments, the red blood cells used in this comparison are human red blood cells. In preferred embodiments, the composition has a P50 of less than 28 mm Hg. In alternative preferred embodiments, the P50 is approximately 1, while in others, it is in the range of 1 to 1.5, while in yet other embodiments, it is approximately 10. However, it is not intended that the present invention be limited to any particular P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.
The other embodiments, the compositions which further comprise a diluent selected from the group consisting of proteins, glycoproteins, polysaccharides, and other colloids. It is not intended that the present invention be limited to any particular diluent. Thus, it is intended that the diluent encompass solutions of albumin, other colloids, or other non-oxygen carrying components. In preferred embodiments, the diluent comprises polysaccharide. In other preferred embodiments, the polysaccharide comprises starch. In particularly preferred embodiments, the starch comprises pentastarch.
In yet other embodiments, the hemoglobin within the composition is surface-modified. It not intended that the present invention be limited to any particular type of surface modification. In preferred embodiments, the surface modification includes the use of polyalkylene oxide groups of varying chain lengths and charge. In preferred embodiments, the hemoglobin is surface-modified with polyethylene glycol of varying chain lengths and charges.
The present invention also provides methods comprising the steps of providing: an organ from an animal, and an aqueous cell-free composition comprising hemoglobin, wherein the hemoglobin is present in a concentration of between 0.1 and 4.0 g/dl, and the aqueous composition has a viscosity that is greater than 2.5 cP; and perfusing the organ with said aqueous composition. In preferred embodiments, the animal is a mammal, while in particularly preferred embodiments, the animal is a human. However, it is not intended that the methods be limited to humans or mammals. In preferred embodiments, the organ is selected from the group consisting of kidneys, liver, spleen, heart, pancreas, lung, and muscle, although it is not intended that the methods of the present be limited to these organs, as any organ may be perfused with the aqueous solution of the present invention.
In some preferred embodiments of the methods, the viscosity of the aqueous composition is between 2.5 and 4 cP. Thus, it is not intended that the present invention be limited to any viscosity that is greater than approximately 2.5 cP. Indeed, it is contemplated that the present invention encompass compositions in which the viscosity is 6 cP or greater. In addition, the present invention encompasses compositions in which the hemoglobin concentration is less than 0.1 or greater than 4 g/dl, although in particularly preferred embodiments, the hemoglobin concentration is between 0.1 and 4 g/dl. In some embodiments, the K* of the composition is approximately equal or similar to that of a red blood cell suspension when measured at the same hemoglobin concentration.
These embodiments also provide compositions comprising hemoglobin with an increased affinity for molecular oxygen as compared to red blood cells. As above, these embodiments are suitable for use in any animal, including humans. Thus, in some embodiments, hemoglobin has an increased affinity as compared to mammalian red blood cells, although in other embodiments, it is contemplated that the red blood cells are from reptiles, avians, or any other animal. In most preferred embodiments, the red blood cells used in this comparison are human red blood cells. In preferred embodiments, the composition has a P50 of less than 28 mm Hg. However, it is not intended that the present invention be, limited to this P50 value, as in some embodiments, the P50 is higher than 28 mm Hg.
The other embodiments, the compositions further comprise a diluent selected from the group consisting of proteins, glycoproteins, polysaccharides, and other colloids. It is not intended that the present invention be limited to any particular diluent. Thus, it is intended that the diluent encompass solutions of albumin, other colloids, or other non-oxygen carrying components. In preferred embodiments, the diluent comprises polysaccharide. In other preferred embodiments, the polysaccharide comprises starch. In particularly preferred embodiments, the starch comprises pentastarch.
In yet other embodiments, the hemoglobin within the composition is surface-modified. It not intended that the present invention be limited to any particular type of surface modification. In preferred embodiments, the surface modification includes the use of polyalkylene oxide groups of varying chain lengths and charge. In preferred embodiments, the hemoglobin is surface-modified with polyethylene glycol of varying chain lengths and charges.
It is not intended that the present invention be limited to any particular oncotic pressure. Indeed, it is intended that the compositions of the present invention encompass a range of oncotic pressure. In some embodiments, the oncotic pressure ranges from 70 to 80 mm Hg, while in the most preferred embodiments, the oncotic pressure is approximately 90 mm Hg. However, in other embodiments, the oncotic pressure can be as low as 60 mm Hg. Furthermore, it is intended that the present invention encompass hypooncotic, hyperoncotic, and isooncotic pressures. As used herein, the term xe2x80x9chyperoncoticxe2x80x9d encompasses any oncotic pressure that is greater than 25 mm Hg, although in preferred embodiments, solutions with oncotic pressures of 20-60 mm Hg are provided. In some embodiments of the methods of the present invention, it is contemplated that the composition chosen for administration will be customized to the particular needs of the animal. The present invention provides the means to customize the composition to meet the needs of various clinical and veterinary uses.
FIG. 19 provides a graph showing the hemoglobin concentration and viscosity of various hemoglobin preparations. The square positioned within this graph (i.e., at approximately 2.5-4 cP and 0.1 to 4 g/dl hemoglobin) indicates the properties of the most preferred compositions of the present invention. As indicated, the only hemoglobin solution that meets the criteria is the xe2x80x9cHemospanxe2x80x9d solution which was made according to the methods of the present invention. The other samples in this graph include blood, PEG-Hb (Enzon), PHP (Apex), and xcex1xcex1-hemoglobin (US Army). As discussed in more detail below, the characteristics of the compositions of the present invention provide many heretofore unknown and unexpected advantages.
The present invention further provides a method comprising: providing i) liganded hemoglobin, ii) means for treating hemoglobin, and iii) means for surface decorating hemoglobin; treating the liganded hemoglobin with the treating means under conditions such that a treated hemoglobin is produced having greater affinity for molecular oxygen than unliganded hemoglobin; and surface decorating the treated hemoglobin with the surface decorating means.
In some embodiments of the method, the means for treating is selected from the group consisting of crosslinking means and polymerizing means. In alternative embodiments, the surface decoration of step (c) comprises reacting said treated hemoglobin with a polyalkylene oxide.
The present invention also provides a method comprising: providing i) liganded hemoglobin, ii) means for treating hemoglobin selected from the group consisting of crosslinking means and polymerizing means, and iii) means for surface decorating hemoglobin; treating the liganded hemoglobin with the treating means under conditions such that a treated hemoglobin is produced having greater affinity for molecular oxygen than unliganded hemoglobin; and surface decorating the treated hemoglobin with the surface decorating means. In some embodiments of the method, the surface decoration of step (c) comprises reacting the treated hemoglobin with a polyalkylene oxide.
The present invention further provides a method comprising: providing i) hemoglobin, ii) means for enzymatically treating hemoglobin (e.g., with enzymes such as carboxy peptidase), and iii) means for surface decorating hemoglobin; treating the liganded hemoglobin with the enzymatic treating means under conditions such that an enzymatically treated hemoglobin is produced having greater affinity for molecular oxygen than hemoglobin in red blood cells; and surface decorating the enzymatically treated hemoglobin with the surface decorating means.
To facilitate uuderstanding of the invention set forth in the disclosure that follows, a number of terms are defined below.
The phrase xe2x80x9coxygen capacity less than that of bloodxe2x80x9d means that when the oxygen capacity of the blood product solutions of the present invention is compared with that of blood, the oxygen capacity of the blood product solutions is less. The oxygen capacity of the blood product solutions of the present invention is not required to be less than that of blood by any particular amount. Oxygen capacity is generally calculated from hemoglobin concentration, since it is known that each gram of hemoglobin binds 1.34 mL of oxygen. Thus, the hemoglobin concentration in g/dL multiplied by the factor 1.34 yields the oxygen capacity in mL/dL. The present invention contemplated the use of a suitable commercially available instruments to measure hemoglobin concentration, including the B-Hemoglobin Photometer (Hemocue, Inc.). Similarly, oxygen capacity can be measured by the amount of oxygen released from a sample of hemoglobin or blood by using, for example, a fuel-cell instrument (e.g., Lex-O2-Con; Lexington Instruments).
The phrase xe2x80x9coxygen affinity equal to or greater than that of bloodxe2x80x9d means that when the oxygen affinity of the blood product solutions of the present invention is compared with that of blood, the oxygen affinity of the blood product solutions is greater. The oxygen capacity of the blood product solutions of the present invention is not required to be greater than that of blood by any particular amount. The oxygen affinity of whole blood (and components of whole blood such as red blood cells and hemoglobin) can be measured by a variety of methods known in the art. (See, e.g., Vandegriff and Shrager in Methods in Enzymology (Everse et al., eds.) 232:460 [1994]). In preferred embodiments, oxygen affinity may be determined using a commercially available HEMOX(copyright) Analyzer (TCS Medical Products). (See, e.g., Winslow et al., J. Biol. Chem., 252(7):2331-37 [1977]).
The phrase xe2x80x9concotic pressure higher than that of plasmaxe2x80x9d means that when the oncotic pressure of the blood product solutions of the present invention is compared with that of plasma, the oxygen affinity of the blood product solutions is greater. The oncotic pressure of the blood product solutions of the present invention is not required to be greater than that of blood by any particular amount. Oncotic pressure may be measured by any suitable technique; in preferred embodiments, oncotic pressure is measured using a Colloid Osmometer (Wesco model 4420).
The phrase xe2x80x9cviscosity at least half of that of bloodxe2x80x9d means that when the viscosity of the blood product solutions of the present invention is compared with that of blood, the oxygen affinity of the blood product solutions is at least 50% of that of blood; in addition, the viscosity may be greater than that of blood. Preferably, viscosity is measured at 37xc2x0 C. in a capillary viscometer using standard techniques. (See Reinhart et al., J. Lab. Clin. Med. 104:921-31 [1984]). Moreover, viscosity can be measured using other methods, including a rotating cone-and-plate viscometer such as those commercially available from Brookfield. The viscosity of blood is approximately 4 centipoise. Thus, at least half of the blood value corresponds to at least approximately 2 centipoise.
The term xe2x80x9cblood productxe2x80x9d refers broadly to formulations capable of being introduced into the circulatory system of the body and carrying and supplying oxygen to the tissues. While the term xe2x80x9cblood productsxe2x80x9d includes conventional formulations (e.g., formulations containing the fluid and/or associated cellular elements and the like that normally pass through the body""s circulatory system, including, but not limited to, platelet mixtures, serum, and plasma), the preferred blood products of the present invention are xe2x80x9cblood product mixtures.xe2x80x9d As used herein, blood product mixtures comprise a non-oxygen-carrying component and an oxygen-carrying component.
The term xe2x80x9coxygen-carrying componentxe2x80x9d refers broadly to a substance capable of carrying oxygen in the body""s circulatory system and delivering at least a portion of that oxygen to the tissues. In preferred embodiments, the oxygen-carrying component is native or modified hemoglobin. As used herein, the term xe2x80x9chemoglobinxe2x80x9d refers to the respiratory protein generally found in erythrocytes that is capable of carrying oxygen. Modified hemoglobin includes, but is not limited to, hemoglobin altered by a chemical reaction such as cross-linking, polymerization, or the addition of chemical groups (e.g., polyethyleneglycol, polyoxyethylene, or other adducts). Similarly, modified hemoglobin includes hemoglobin that is encapsulated in a liposome.
The present invention is not limited by the source of the hemoglobin. For example, the hemoglobin may be derived from animals and humans; preferred sources of hemoglobin are cows and humans. In addition, hemoglobin may be produced by other methods, including recombinant techniques. A most preferred oxygen-carrying-component of the present invention is xe2x80x9cpolyethylene glycol-modified hemoglobin.xe2x80x9d
The term xe2x80x9cpolyethylene glycol-modified hemoglobinxe2x80x9d refers to hemoglobin that has been modified such that it is associated with polyethylene glycol (xcex1-Hydro-xcfx89-hydroxypoly-(oxy-1,2-ethanediyl); generally speaking, the modification entails covalent binding of polyethylene glycol (PEG) to the hemoglobin. PEGs are liquid and solid polymers of the general chemical formula H(OCH2CH2)nOH, where n is greater than or equal to 4. PEG formulations are usually followed by a number that corresponds to its average molecular weight; for example, PEG-200 has a molecular weight of 200 and a molecular weight range of 190-210. PEGs are commercially available in a number of formulations (e.g., Carbowax Poly-G, and Solbase).
The term xe2x80x9cnon-oxygen-carrying componentxe2x80x9d refers broadly to substances like plasma expanders that can be administered, e.g., for temporary replacement of red blood cell loss. In preferred embodiments of the invention, the non-oxygen-carrying component is a colloid (i.e., a substance containing molecules in a finely divided state dispersed in a gaseous, liquid, or solid medium) which has oncotic pressure (colloid osmotic pressure prevents, e.g., the fluid of the plasma from leaking out of the capillaries into the interstitial fluid). Examples of colloids include hetastarch, pentastarch, dextran-70, dextran-90, and albumin.
Preferred colloids of the present invention include starches like hetastarch and pentastarch. Pentastarch (hydroxyethyl starch) is the preferred colloid starch of the present invention. Pentastarch is an artificial colloid derived from a starch composed almost entirely of amylopectin. Its molar substitution is 0.45 (i.e., there are 45 hydroxyethyl groups for every 100 glucose units); hydroxyethyl groups are attached by an ether linkage primarily at C-2 of the glucose unit (and less frequently at C-3 and C-6). The polymerized glucose units of pentastarch are generally connected by 1-4 linkages (and less frequently by 1-6 linkages), while the degree of branching is approximately 1:20 (i.e., there is one branch for every 20 glucose monomer units). The weight average molecular weight of pentastarch is about 250,000 with a range of about 150,000 to 350,000. Unless otherwise indicated, reference to the xe2x80x9caverage molecular weightxe2x80x9d of a substance refers to the weight average molecular weight. Pentastarch is commercially available (e.g., DuPont Merck) as a 10% solution (i.e., 10 g/100 mL); unless otherwise indicated, reference to blood product solutions comprising pentastarch (and other non-oxygen-carrying components as well as oxygen-carrying components) is on a volume basis.
The phrase xe2x80x9cenhancing oxygen delivery to the tissues of a mammalxe2x80x9d refers to the ability of a fluid (e.g., a blood product) introduced into the circulatory system to deliver more oxygen to the tissues than would be delivered without introduction of the fluid. To illustrate, a patient may experience substantial blood loss following acute hemorrhage, resulting in decreased transport of oxygen to the tissues via the blood. The administration of a blood product to the patient can supplement the ability of the patient""s own blood to deliver oxygen.
The term xe2x80x9cmixturexe2x80x9d refers to a mingling together of two or more substances without the occurrence of a reaction by which they would lose their individual properties. The term xe2x80x9csolutionxe2x80x9d refers to a liquid mixture. The term xe2x80x9caqueous solutionxe2x80x9d refers to a solution that contains some water. In many instances, water serves as the diluent for solid substances to create a solution containing those substances. In other instances, solid substances are merely carried in the aqueous solution (i.e., they are not dissolved therein). The term aqueous solution also refers to the combination of one or more other liquid substances with water to form a multi-component solution.
The term xe2x80x9capproximatelyxe2x80x9d refers to the actual value being within a range of the indicated value. In general, the actual value will be between 10% (plus or minus) of the indicated value.